a-Melanocyte-stimulating hormone (a-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of a-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and a-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon Y.a-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. a-MSH increased cAMP accumulation in RAW cells, characteristic of a-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary c-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecule of a-MSH, was expressed in RAW cells, and tumor necrosis factor a increased production and release of cv-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor a can induce macrophages to increase production of c-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that a-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines.The neuropeptide a-melanocyte-stimulating hormone (a-MSH), a derivative of proopiomelanocortin (POMC), found in the pituitary, brain, skin, circulation, and other sites, has potent antiinflammatory activity. a-MSH reduces fever (1, 2) and the following: (i) acute inflammation caused by irritants or by local mediators, such as cytokines (3, 4); (ii) delayed hypersensitivity responses, a model of allergic reactions (5, 6); (iii) chronic inflammation (mycobacterium-induced arthritis) (7,8); and (iv) systemic inflammation (e.g., endotoxemia and sepsis) (8, 9). Although a-MSH antagonizes the actions of proinflammatory cytokines (1, 2, 4, 9, 10), the precise mechanism of its antiinflammatory action is unknown. a-MSH was recently identified in synovial fluid of patients with rheumatoid arthritis (11). Thus, a-MSH is produced at sites of inflammation, although the cell type(s) responsible is unknown.a-MSH functions via melanocortin (MC) receptors (12-18). There are five subtypes of MC receptors (MC-1 through MC-5). These receptors contain seven membrane-spanning domains typical of G-protein-coupled receptors, and are coupled to adenylate cyclase to generate cAMP. MC receptors are differentially expressed in a restricted distribution, with MC-1 identified in melanoma cells and melanocytes but not in brain, adrenal, or other tissues by mRNA blotting (12, 13).That a-MSH has a wide spectrum of antiinflammatory activity suggests that the peptide inhibits a critical agent or event that is common to multiple forms of inflammatio...

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alpha-Melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide derived from pro-opiomelanocortin, has potent antiinflammatory activity in laboratory animals. alpha-MSH inhibits nitric oxide production by murine macrophages, an influence believed to reflect activation of an autocrine circuit in these cells, one that is based on production and release of alpha-MSH and subsequent stimulation of melanocortin receptors. We found that THP-1 cells, human monocytic cells, produced alpha-MSH; this production was increased by interleukin-6, tumor necrosis factor a, or concanavalin A. These cells also expressed the gene for the human alpha-MSH receptor MC1. Unlike murine macrophages, THP-1 cells produced little nitrite in response to interferon-gamma (IFN-gamma) and lipopolysaccharide, and a-MSH inhibited this production only slightly. However, production of neopterin, a presumed primate homologue of nitric oxide in lower animals, was increased in THP-1 cells stimulated with INF-gamma plus TNF-alpha and alpha-MSH significantly inhibited this production. The evidence indicates that an autocrine regulatory circuit based on alpha-MSH occurs in human monocyte/macrophages much as in murine macrophages. alpha-MSH-induced modulation of specific inflammatory mediators/cytotoxic agents appears to differ depending on the importance of the mediators in the myelomonocytic cells of different species.

The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and α-melanocyte-stimulating hormone (α-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of α-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-α was inhibited in relation to α-MSH concentration. Similar inhibitory effects on TNF-α were observed with ACTH peptides that contain the α-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-α concentration; the antibody also markedly reduced the inhibitory influence of α-MSH on TNF-α in macrophages treated with LPS. These results in cells known to produce α-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

Tumor necrosis factor (TNF-α) underlies pathological processes and functional disturbances in acute and chronic neurological disease and injury. The neuroimmunomodulatory peptide α-MSH modulates actions and production of proinflammatory cytokines including TNF-α, but there is no prior evidence that it alters TNF-α induced within the brain. To test for this potential influence of the peptide, TNF-α was induced centrally by local injection of bacterial lipopolysaccharide (LPS). α-MSH given once i.c.v. with LPS challenge, twice daily intraperitoneally (i.p.) for 5 d between central LPS injections, or both i.p. and centrally, inhibited production of TNF-α within brain tissue. Inhibition of TNF-α protein formation by α-MSH was confirmed by inhibition of TNF-α mRNA. Plasma TNF-α concentration was elevated markedly after central LPS, indicative of an augmented peripheral host response induced by the CNS signal. The increase was inhibited by α-MSH treatments, in relation to inhibition of central TNF-α. Presence within normal mouse brain of mRNA for the α-MSH receptor MC-1 suggests that the inhibitory effects of α-MSH on brain and plasma TNF-α might be mediated by this receptor subtype. The inhibitory effect of α-MSH on brain TNF-α did not depend on circulating factors because the effect also occurred in brain tissuein vitro. This indicates that α-MSH can act directly on brain cells to inhibit their production of TNF-α. If central TNF-α contributes to pathology in CNS disease and injury, and promotes inflammation in the periphery, agents that act on brain α-MSH receptors should decrease the pathological TNF-α reaction and promote tissue survival.